Use of a polyfunctional active substance mixture as an antagonist against harmful substances contained in tobacco smoke

ABSTRACT

The invention relates to the use of a polyfunctional active substance mixture with anti-inflammatory, spasmolytic and anti-stress action comprised of a fraction of specific peptides with molar weights of up to 10,000 dalton and/or of a fraction of essential and non-essential amino acids, and the polyfunctional active substance mixture is obtained from a multifactorial immune-modulator mixture serving as an antagonist against the harmful substances contained in tobacco smoke with a health-protecting function during the smoking of tobacco products. It is preferably applied in the form of an inhalant (adhesion principle, atomization) and leads to the amelioration, elimination and prevention of inflammations of the mucous membranes, of regulatory disorders of the organ systems with smooth muscles (blood vessels, bronchial system, esophagus, bladder, stomach, intestines, etc.), of the myocardial function as well as of the central and peripheral nerve systems that are induced by the harmful substances contained in tobacco smoke.

The invention relates to the use of a polyfunctional active substancemixture having anti-inflammatory, spasmolytic and anti-stress actioncomprised of a fraction of specific peptides with molar weights of up to10,000 dalton and of a fraction of essential and non-essential aminoacids, and whereby the mixture was obtained from a multifactorialimmune-modulator mixture and is suitable for use as an antagonistagainst harmful substances contained in tobacco smoke and having ahealth-protecting function during the smoking of tobacco.

The mixture is preferably applied as an inhalant (adhesion principle,atomization) and results in the amelioration, elimination and preventionof inflammations induced by the harmful substances contained in tobaccosmoke of the mucous membranes, of regulatory disorders of the organsystems with smooth muscles (including blood vessels, bronchial system,esophagus, bladder, stomach, intestines, etc.), of the myocardialfunction as well as of the central and peripheral nervous system. Thepolyfunctional active substance mixture is obtained from amultifactorial immune-modulator blend.

The number of scientific works addressing the harmful effects thattobacco smoking has on health and the pathogenic effects of tobaccosmoking is endless. This has led to a situation in which governments andcourts must now deal with this problem and will continue to have to dealwith it. We are presently listing only some of the examples.

-   -   1. EU-Commissioner for Health and Consumer Protection, David        Byrne, (Focus 11/2000, p. 362/363), “Half a million EU citizens        die annually as a consequence of the consumption of tobacco—this        is one death per minute.”    -   2. The World Health Organization is negotiating an anti-tobacco        convention (same source).    -   3. The smoking of pregnant mothers increases prenatal stresses        with carcinogenic and teratogenic substances (Deutsches        Ärzteblatt [German Physicians' Gazette] 97/39 2000, p. C 1873).    -   4. In a jury trial in the United States, the US tobacco industry        was sentenced to pay $145 million to sick smokers. This is the        highest judgment ever achieved for damages in a civil case in        the United States (Focus 51/2000, p. 168).        A Brief Summary of the Essential Pathogenic Consequences of        Tobacco Smoking

1. It is known that tobacco smoking, primarily cigarette smoking, hasharmful effects due to the harmful substances contained therein whichcause chronic inflammations of the mucous membranes of the digestive andrespiratory systems as well as of the eyes. Primarily affected are themucous membranes of the mouth, the nose, the throat and the larynx, thebronchial system and the bronchioles, the esophagus, the stomach, theduodenum, the small intestine and the large intestine. Many peoplesuffer from chronic conjunctivitis due to cigarette smoking. Theseinflammations that are caused by the harmful substances in tobacco smokefrequently lead to ulcerations in the digestive tract, the complete orpartial inhibition of the resorption of vitamins C and betacarotene—vitamin A —(which may cause diseases specific to a vitamindeficiency), infectious diseases of the respiratory organs, etc.;finally, the ultimate consequence of this is a high incidence ofhealth-related costs (refer to litigation in the United States).

-   -   2. It is known that the harmful substances contained in tobacco        smoke (approximately 200 harmful substances have been detected)        result in inflammations of the mucous membranes, disorders of        the smooth muscles presenting as contraction or spasm of the        blood vessels, in the bronchial system and in the digestive        system. Deficient blood supply to the heart (myocardial        infarction), to the skin, the brain, the extremities (smoker's        leg, etc.), and primarily chest-related complaints of the smoker        are the most common disorders, as reflected in the pertinent        statistics.    -   3. It is known that substances contained in tobacco smoke also        act as stress factors and, e.g., cause adrenalin, the adrenal        medullary hormone, to be increasingly released into the blood        stream triggering generalized distress. This causes, on the one        hand, the pathological manifestations listed under 1. and 2. to        be stimulated and thereby to become enhanced, and triggers or        causes, on the other hand, the regulatory disorders that are        known consequences of distress, e.g., of the immune system and        the transmitter system (including regulatory peptides and        neuro-peptides).    -   4. It is known today that smoking constitutes an addiction and        that quitting is usually not only difficult but also accompanied        by many severe pathological withdrawal symptoms. This is why        many smokers continue to smoke even though they are familiar        with the health-related risks.    -   5. The logical conclusion that is drawn based on these        scientific findings is that smoking is harmful to a person's        health and that every pack of cigarettes or related advertising        must contain a warning of these harmful effects.    -   6. The tobacco smoker is addicted and intends to continue        smoking because the smoker is addicted even though the smoker is        aware of the fact that he/she is harming his/her health. This        leads to a constant psychological conflict, often also taking        place on an unconscious level.    -   7. A substance that protects against the health-related damage        due to tobacco smoking has not been available to date, even        though such a substance would be urgently indicated and of great        usefulness in order to ease, first, the stress of the internal        conflict that the already addicted smoker is struggling with        and, second, to protect the already addicted smoker from        additional health-related harm of tobacco smoking.

Therefore, the object of the present invention consisted in identifyingan antagonist against the harmful substances contained in tobacco smokethat has a health-protecting function and that on its part does not posea health risk or cause addiction, that does not compromise the taste ofthe tobacco product and the smoking enjoyment, that is easily usable andthat serves to ameliorate, eliminate and prevent tobacco smoke induced

-   -   inflammations of the mucous membranes;    -   regulatory disorders;    -   the function of the organ systems with smooth muscle (bronchial        system, blood vessels, esophagus, bladder, stomach, intestines,        etc.);    -   the cardiac function; and,    -   the functions of the central and peripheral nerve systems.

It was possible to achieve this object with a polyfunctional activesubstance mixture that is characterized by its anti-inflammatory,spasmolytic and anti-stress action and that is comprised of a fractionof specific peptides with molar weights of up to 10,000 dalton and/or ofa fraction of essential and non-essential amino acids obtained from amultifactorial immune-modulator blend.

In terms of its application, the polyfunctional active substance mixtureis preferably incorporated with the tobacco product and administered tothe body as an inhalant (adhesion principle, atomization via respiratorypathways and respiratory organs) by way of a mouth tip filter of thetobacco product (cigarette, cigar, cigarillo, tobacco pipe).

Multifactorial immune-modulator mixtures with anti-inflammatory actionas such are known in the art. A modulator mixture that is essentiallydescribed in DE 195 12 227 C1 is used; but said mixture differs from theblend in DE 195 12 227 C1 in that it represents a fraction of specificpeptides with a molecular weight of up to 10,000 dalton and/or of afraction of essential and non-essential amino acids. It is synthesizedanalogously to DE 195 12 227 C1, i.e., it is obtained from somatic cellsand, in contrast to DE 195 12 227C1, the fraction of specific peptideswith a molecular weight of up to 10,000 dalton and/or containingessential and non-essential amino acids is isolated by way of high-speedcentrifuging at a running time of 24 hours. This means thepolyfunctional active substance mixture with anti-inflammatory,spasmolytic and anti-stress action comprised of a fraction of specificpeptides with a relative molar mass of up to 10,000 dalton and/or ofessential and non-essential amino acids [is obtained] from the somaticcells by way of

-   -   incubation at temperatures that are typical for the original        species in the range of 20 to 39° C. for a time period of 2 to 8        days;    -   followed by lysis;    -   harvested together with the conservation medium; and,    -   separation of the components having a molar mass of over 10,000        dalton and simultaneous isolation of the fraction of specific        peptides with molar weights of up to 10,000 and/or of the        fraction of essential and non-essential amino acids by way of        high-speed centrifuging at a running time of 24 hours.    -   A polyfunctional active substance mixture containing a fraction        of specific peptides with a relative molar mass of between 200        and 6,000 dalton is preferred. This mixture was achieved via        reconcentration by a factor of 1,000 of the obtained fractions        with proteins <10,000 dalton and using physical means. The        content materials were analyzed by way of mass spectroscopy.

These peptides <10,000 dalton that are contained in the mixture arepeptides that also occur in the human organism, which is why an“endogenic” active substance mixture is available that is welltolerated. Endogenic peptides are widely known and have been described.Within the meaning of the present invention, preferably those peptidesare active within the polyfinctional mixture of active substances thatare to be described as acting in the way of a homeostasis regulator,stress regulator, as acting upon the smooth muscles and/or asneuro-peptides (Hecht, K., W-E Vogt, E. Wachtel, P. Oehme and M.G.Airapetjanz: Sleep-Regulating Peptides. Beiträge zur Wirkstoffforschung[Contributions to the Active Substance Research]. Vol. 37 dito, 1990),(Oehme, P., H. Bienert, K. Hecht, J. Bergmann: Substance P. Beiträge zurWirkstoffforschung [Contributions to the Active Substance Research].Vol. 12 dito, 1981), (Marsan, C. A., W. Z. Trazyk (eds.): Neuropeptidesand Neural Transmission. International Brain Research Organization(IBRO) Monograph Series: Volume 7. Raven Press New York), Oehme, P., H.Löwe, E. Göres (eds.): Peptide and Adaptation Basic Research andClinical Aspects. Beiträge zur Wirkstoffforschung [Contributions to theActive Substance Research], Vol. 36, Academic-Industrial ComplexPharmaceutical Research Berlin (1990), Oehme, P., H. Löwe, E. Göres(eds.): Peptides in the Nervous System. Beiträge zur Wirkstoffforschung[Contributions to the Active Substance Research], Vol. 24 (1985).

A health-protecting antagonist against tobacco smoke was found in thispolyfunctional active substance mixture that is preferably introduced asan inhalant (adhesion principle, atomization) by way of a mouth tipfilter on the tobacco product (cigarette, cigar, cigarillo, tobaccopipe, etc.) and thereby administered to the body.

For this purpose, the polyfunctional active substance mixture isintegrated into the mouth piece (filter) of the tobacco product(cigarette, cigar, cigarillo, tobacco pipe etc.) as part of themanufacturing process in the form of a substrate that is to be atomized,and that is in fact atomized in accordance with the adhesion principle,while smoking; thereby it is blended in with the tobacco smoke andintroduced into the body.

The simultaneous application with the tobacco product does in no waycompromise the tobacco taste for the smoker. The polyfunctional activesubstance mixture has moreover no toxically relevant effect. It developsrelevance for better health even at very low concentrations (10⁻⁶ to 10⁻⁹ g/kg of body weight), which means it is efficacious via a bioactivetrigger function. The polyfunctional active substance mixture hasspecific effective properties that are to be described asanti-inflammatory, promoting blood flow, stimulating resorption andpermeability, relaxing spasms, immuno-modulating, reducing distress,stimulating eustress and regulating homeostasis. The dose-actionrelationship preferably follows the principle of the hyperbola curve.After a completed bioactive trigger function effect, it is quicklymetabolized in the body, which means that overdoses are not possibleeven in heavy smokers. The development of any resistance vis-a-vispathogenic organisms was not shown.

The substance leads to an amelioration, elimination and prevention ofinflammations of the mucous membranes, of regulatory disorders of thesmooth muscle organ systems (blood vessels, bronchial system, esophagus,bladder, stomach, intestines, etc.) of the myocardial function as wellas of the central and the peripheral nerve systems that are induced bythe harmful substances contained in tobacco smoke.

Using these example, the invention will be illustrated in more detail:

EMBODIMENTS

1. Synthesis of the Mixture

EXAMPLE 1

The diploid calf kidney cell line MDBK (ATCC no. CCL 22) is,corresponding to its multiplication rate of 1:4 in DMEM, added to 8%newborn calf serum and then incubated inside roller bottles at 38° C.Larger quantities are, following the adaptation of the cells, morefavorably bred inside spinning culture vessels or inside fermenters.After 3 to 4 days the culture has optimally completed its growth phase.The breeding medium is removed and replaced with MEM without serum. Thecell cultures are incubated for an additional 5 to 7 days. The longerthe incubation time, the more the intracellular pool is deprived of itsessential nutrients; mediators and low-molecular metabolic end productsare found at increasing concentrations in the MEM. The accumulation ofmetabolic products and the intracellular lack of essential nutrients isultimately a stress factor for the aging cell culture. The cell culturereacts to this situation with the synthesis of various “stressproteins.” These stress proteins become concentrated inside the cell.The cell cultures are regularly examined under a light microscope duringthe concentration phase. No cytopathic changes may be visible after theconcentration phase is concluded. The concentration phase is finishedwith the alkaline purification of the MEM using NaOH and adjustment to apH 10 as well as a temperature increase to 50° C. for the duration of 60minutes. Instead of shifting the pH value, freezing of the cells hasalso proved effective. Freezing occurs favorably at −20° C. After thattime, the died-off cells can be easily shaken off the surface of theculture vessel. Temperature increase and pH-shift do not influence thebiological activity of the modulator mixture. The conservation mediumwith the extracellular and intracellular modulators is now freed of celldetritus and particulate components by way of step filtration. Theobtained filtrate contains the mixture of modulators, which are freedfor the most part of membrane components. Sterile storage without lossof activity is possible for several weeks at 4° C. or by freezing (−20°C.) for at least one year.

By way of ultrafiltration with commonly used instruments, and while thepore size of the filtration membranes holds back globular molecules,fractions with a relative molar mass starting at approximately 10,000[dalton] are retained. The retained substance contains all extracellularand intracellular materials with a relative molar mass for globularproteins that exceeds 10,000 [dalton] and particles with diameters ofless than 9 nm.

The polyfunctional active substance mixture according to the inventioncomprised of the fraction of specific peptides up to a molar weight of10,000 dalton and of essential and non-essential amino acids wasobtained by ultracentrifuge with a running time of 24 hours from theprotein mixture (analogous to DE 195 12 227 C1 of Oct. 3, 1996). Thefractions with materials of up to 10,000 dalton molar weight werereconcentrated by a factor of 1,000 and subsequently analyzed by way ofmass spectroscopy to determine their content materials. The obtainedspectrum documented a particularly high level of materials with molarweights of between 200 and 6,000 dalton.

The polyfunctional active substance mixture that is obtained in this waycan be stored without loss of activity for several weeks at 4° C. orfrozen (−20° C. and lower) for at least one year. It can also belyophilized in the usual manner. The purity, harmlessness and efficacyof the modulator mixture according to the invention is verified by wayof the corresponding control testing. The purity and harmlessness checksare conducted in accordance with the prescribed methods of the EuropeanPharmacopoeia and/or the German Pharmacopoeia [DAB=DeutschesArzneimittelbuch] 10. Protein determination and polyacryl gelelectrophoresis are conducted using the long known standard protocols.

Purity

-   -   1. Testing for sterility in accordance with the European        Pharmacopoeia/German Pharmacopoeia 10;    -   2. Determination of the protein content in accordance with        Lowry;    -   3. Separation and quantification of the main proteins by way of        polyacrylamide gel electrophoresis under denaturing conditions.        Harmlessness    -   1. Testing for the absence of pyrogene in accordance with the        European Pharmacopoeia/German Pharmacopoeia 10;    -   2. Testing for acute toxicity in accordance with the European        Pharmacopoeia/German Pharmacopoeia 10;    -   3. Testing for teratogeneity in accordance with the European        Pharmacopoeia/German Pharmacopoeia 10.

A typical preparation is sterile, free of pyrogene, non-toxic andnon-teratogenic. It contains approximately 2 to 3 mg protein per ml.

EXAMPLE 2

Chicken fibroblasts, obtained by way of typtic cell isolation of chickenembryos, are cultivated in accordance with example 1 in roller bottles.Reconcentration, harvesting and treatment as well as testing foridentity, harmlessness, and efficacy was conducted in correspondencewith example 1. Achieved are essentially identical results to example 1.

EXAMPLE 3

A diploid, limited growth tube epithelium cell line of the pig (Latzke,1993) underwent stationary cultivation in accordance with example 1.Reconcentration, harvesting and treatment as well as testing foridentity, harmlessness, and efficacy was conducted in correspondencewith example 1.

2. Application Examples

EXAMPLE 4

In the private practice of a general medical practitioner of alternativemedicine, 22 patient volunteers suffering [respectively] from an acuteinfection of the respiratory pathways were administered the describedpolyfunctional active substance mixture in the form of an one-timeinhalant. Within 24 hours, the symptoms were no longer detectable. Incontrast, in patients (n=28) who presented with the same symptoms at thesame time and who were treated with the conventional therapeutic means,this disease process lasted 3 to 7 days.

EXAMPLE 5—SINGLE CASES

-   -   5.1 Bronchial pneumonia: A 48-year-old female patient volunteer        who had been suffering from bronchitis and an undiagnosed        bronchial pneumonia was treated with the described        multifunctional active substance mixture in the form of an        inhalant administered twice daily. As early as after two        inhalations, the symptoms started to subside. Within a week, the        symptoms had for the most part disappeared.    -   5.2 Viral bronchitis: A 76-year-old male patient had been        suffering for approximately 2 weeks from a therapy-resistant        chronic viral bronchitis accompanied by strong nightly attacks        of a hacking, dry cough. After the first inhalation of the        described polyfunctional n active substance mixture, the nightly        hacking cough eased up; after two inhalations the entire        bronchitis disappeared.    -   5.3: A 76-year-old male patient had been suffering for years        from frequently occurring nose bleeds (apparently of an allergic        nature). After eight inhalations of the described polyfunctional        n active substance mixture administered twice daily, the nose        bleeds did not return. The follow-up control period is currently        6 months.

EXAMPLE 6—HYPERFUNCTION SMOKING, ACTIVE SUBSTANCE INHALATION

The efficacy of the described polyfunctional active substance mixturewas tested in 12 volunteers while they were each smoking a cigaretteusing a very sensitive heart function warning system that operated onECG basis (Jumatow Moskow). One time the cigarette was smoked withoutthe polyfunctional active substance mixture and one time with themixture. While the heart function warning system signaled the risk whenthe cigarettes without active substance inhalant were smoked, thewarning was not triggered when the atomized active substance mixture wasinhaled simultaneously with the cigarette smoke.

1. Use of a polyfunctional active substance mixture withanti-inflammatory, spasmolytic and anti-stress action as an antagonistagainst the harmful substances contained in tobacco smoke comprised of afraction of specific peptides with molar weights of up to 10,000 daltonand/or of a fraction of essential and non-essential amino acids, andsaid polyfunctional active substance mixture was obtained from amultifactorial immune-modulator mixture: by way of incubation at thetemperatures that are typical for the original species of 20 to 39° C.for a time period of 2 to 8 days; followed by lysis; harvested togetherwith the conservation medium; and, separation of cell components whichhave a molar mass of over 10,000 dalton by way of high-speedcentrifuging at a running time of 24 hours; and, gaining the fraction ofspecific peptides with molar weights of up to 10,000 dalton and/or ofthe fraction of essential and non-essential amino acids.
 2. Use asclaimed in claim 1 wherein a fraction of specific peptides with molarweights of between 200 and 6,000 dalton and/or a fraction of essentialand non-essential amino acids are applied.
 3. Use as claimed in claim 1wherein mixtures of peptides, as well as neuro-peptides containedtherein, are applied that regulate homeostasis, act upon the smoothmuscles and regulate stress.
 4. Use as claimed in one of the claims 1 to3 wherein the mixture is applied at low concentrations, preferably at adosage of 10⁻⁶ to 10⁻⁹ g/kg of body weight and wherein its action occursprimarily in the form of a bioactive triggering function.
 5. Use asclaimed in one of the claims 1 to 4 wherein the dose-action relationshipfollows the principle of a hyperbolic curve whereby an overdose isimpossible.
 6. Use as claimed in one of the claims 1 to 5 wherein themixture is applied in a specific filter (specific mouth tip) of thetobacco product (cigarette, cigar, cigarillo, tobacco pipe, etc.) andwherein the mixture is incorporated into the tobacco product at the timeof the manufacture of the tobacco product and inhaled during smoking. 7.Use as claimed in one of the claims 1 to 6 wherein it [the filter]itself is not harmful to the smoker's health, nor does it constitute arisk for addiction, and it does not change the smoking enjoyment.
 8. Useas claimed in one of the claims 1 to 7 wherein the polyfunctional activesubstance mixture is applied for ameliorating, eliminating andpreventing: inflammations of the mucous membranes; regulatory disorders;[impairment] of the function of the organ systems with smooth muscles(bronchial system, blood vessels, esophagus, bladder, stomach,intestines, etc.); [impairment] of the myocardial functions; and,[impairment] of the functions of the central and peripheral nerve systeminduced by the harmful substances contained in tobacco smoke. 9.Specific filters for tobacco products wherein these filters comprise apolyfunctional active substance mixture as claimed in one of the claims1 to 4 featuring a health-protecting function during the smoking oftobacco.